Composition of polyethylene glycol maleimide derivative and polymerization inhibitor

ABSTRACT

The present invention provides a composition of a polyethylene glycol maleimide derivative and a polymerization inhibitor. In particular, the present invention provides a composition of an 8-arm polyethylene glycol maleimide derivative and a phenolic polymerization inhibitor. The ingredient and content of the polymerization inhibitor in the composition are reasonably chosen, thereby significantly increasing stability of the polyethylene glycol maleimide derivative, effectively avoiding the undesirable effect of gel solidifying due to polymerization during storage and transportation, and extending a pot life and shelf life of a product thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.16/588,246, filed on Sep. 30, 2019, which is a continuation ofInternational Application No. PCT/CN2018/077231, filed on Feb. 26, 2018.The International Application claims priority to Chinese patentapplication No. CN201710209106.6 filed on Mar. 31, 2017 andCN201710530958.5 field on Jun. 30, 2017 respectively. All of theaforementioned applications are hereby incorporated by reference intheir entireties.

TECHNICAL FIELD

The invention relates to the technical field of polymers, in particularto a composition of a polyethylene glycol maleimide derivative and apolymerization inhibitor, in more particular to an eight-armpolyethylene glycol maleimide derivative and a phenol. A composition ofa class of polymerization inhibitors.

BACKGROUND TECHNIQUE

Polyethylene glycol (PEG) is a non-toxic, amphiphilic macromolecularcompound formed by polymerization of ethylene glycol monomers. PEGmodification is a technology developed from the late 1970s. To solvemany problems in the clinical application of some drugs such as peptidesand protein drugs, some PEG modified products have achieved good resultswhen used in a drug. The hydroxyl group at the end of the polyethyleneglycol is a functional group for its chemical reaction, but thereactivity is poor, and a derivative of polyethylene glycol which isactivated in an appropriate way is often used as a modifier. Theactivated terminal groups such as amino, carboxyl, aldehyde, maleimideand the like play a decisive role in the application of polyethyleneglycol. Different terminal groups have different uses, and theintroduction of these reactive groups expands the application range ofPEG. For polyethylene glycol maleimide derivative (PEG-MAL), a maleimidegroup is introduced into PEG, and the coupling of maleimide andsulfhydryl group is one of useful reactions to couple protein andpolypeptide. PEG-MAL can be used as a polymer reagent to selectivelytrap thiol-containing peptides. Sulfhydryl group(s) can be introducedinto specific sites of a peptide and a protein by geneticallyengineering, and PEG-MAL can be further used for site-specificmodification. The selectivity of the modification is high, and the lossof biological activity of the protein could be avoided, and in themeanwhile the immunogenicity could be lowered. In recent years, it hasbeen found that PEG-MAL plays a key role in the linkage between proteinsor peptides and liposomes. PEG-MAL is of great significance fortargeting liposomes and for expanding the application of peptidecompounds in medicine and biotechnology. Therefore, PEG-MAL is apolyethylene glycol derivative with a very high application value.

However, the maleimide derivative of polyethylene glycol contains anunsaturated double bond which has a high reaction activity, resulting inpoor stability of the derivative, and polymerization at room temperatureoften occurs at room temperature to form a gel-like insoluble matter.This would cause the content of the derivative to be low, and the shelflife is short, leading to great inconvenience to its preservation andtransportation, thereby limiting its application.

SUMMARY OF THE INVENTION

The inventors of the present application have found through extensiveexperiments and studies that a polyethylene glycol maleimide derivative(especially an eight-arm polyethylene glycol maleimide derivative) andsome polymerization inhibitors can be combined to enhance its stabilityor reduce its photosensitivity, extending product pot life and shelflife.

In one aspect, the invention provides a composition of a polyethyleneglycol maleimide derivative and a polymerization inhibitor.

In the composition, the mass ratio of the polymerization inhibitor tothe polyethylene glycol maleimide derivative is ≥0.1 μg:1 g.

In one embodiment of the present invention, the mass ratio of thepolymerization inhibitor to the polyethylene glycol maleimide derivativemay be from 0.1 μg to 10 mg:1 g, such as 0.1 to 100 μg.:1 g (eg, 0.1μg:1 g, 1 μg:1 g, 5 μg:1 g, 10 μg:1 g, 20 μg:1 g, 30 μg:1 g, 40 μg:1 g,50 μg:1 g, 60 μg, 70 μg:1 g, 80 μg:1 g, 90 μg:1 g or 100 μg:1 g), 100μg-1 mg:1 g (eg, 100 μg:1 g, 200 μg:1 g, 300 μg:1 g, 400 μg:1 g, 500μg:1 g, 600 μg:1 g, 700 μg:1 g, 800 μg:1 g, 900 μg:1 g or 1 mg:1 g),1-10 mg:1 g (eg, 1 mg:1 g, 2 mg:1 g, 3 mg:1 g, 4 mg:1 g, 5 mg:1 g, 6mg:1 g, 7 mg:1 g, 8 mg:1 g, 9 mg:1 g or 10 mg:1 g); preferably 1 μg to 1mg:1 g.

In the composition, the polyethylene glycol maleimide derivativecontains at least one terminal maleimide group.

In one embodiment of the invention, the polyethylene glycol maleimidederivative has the following structure:PEG-X-MAL   (I)

Wherein PEG is a polyethylene glycol residue,

X is a linking group of PEG and MAL, and is selected from: one or acombination of two or more of —(CH₂)_(r)—,

—(CH₂)_(r)O—, —(CH₂)_(r)CO—, —(CH₂)_(r)NH—, —(CH₂)_(r)CONH—,—(CH₂)_(r)NHCO—, —(CH₂)_(r)S—, —(CH₂)_(r)COO— and —(CH₂)_(r)OCO—, r isan integer from 0 to 10, MAL is a maleimide group

R₁ and R₂ are independently selected from the group consisting of: —H, aC1-6 alkyl group, a C1-6 alkoxy group, a C3-6 cycloalkyl group, and aC4-10 alkylene cycloalkyl group.

Preferably, said R₁ and R₂ are independently selected from the groupconsisting of: —H, —CH₃, —CH₂CH₃, —CH₂CH₃, —CH₂CH₂CH₃, —OCH₃, —OCH₂CH₃and —OCH₂CH₂CH₃, more preferably from: —H, —CH₃, —OCH₃ and —OCH₂CH₃; Ina preferred embodiment of the invention, R₁ is H, R₂ is —CH₃, —OCH₃ or—OCH₂CH₃; in a more preferred embodiment of the invention, R₁ is H andR₂ is —CH₃.

In a specific embodiment of the invention, the X is selected from thegroup consisting of: one or a combination of two or more of a singlebond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂—,—CH(CH₃)—, —CH₂CH(CH₃)—, —CH₂CH₂CH(CH₃)—, —CH₂CH₂CH₂CH(CH₃)—,—CH₂CH₂CH₂CH₂CH(CH₃)—, —CH₂CH₂CH₂CH₂CH₂CH(CH₃)—, —(CH₂)_(r)O—,—(CH₂)_(r)CO—, —(CH₂)_(r)NH—, —(CH₂)_(r)CONH—, —(CH₂)_(r)NHCO—,—(CH₂)_(r)S—, —(CH₂)_(r)COO— and —(CH₂)_(r)OCO—.

In a preferred embodiment of the invention, the X is selected from thegroup consisting of: one or a combination of two or more of a singlebond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH(CH₃)—, —CH₂CH(CH₃)—,—CH₂CH₂CH(CH₃)—, —(CH₂)_(r)CO—, —(CH₂)_(r)NH—, —(CH₂)_(r)CONH— and—(CH₂)_(r)NHCO—.

In one embodiment of the invention, r is an integer from 0 to 5, such as0, 1, 2, 3, 4 or 5.

In a more preferred embodiment of the invention, the X is—CH₂CH₂NHCOCH₂CH₂—.

In the composition of the present invention, the PEG may be a linear, Y-or multi-branched polyethylene glycol residue, for example, including alinear polyethylene glycol, a Y- or U-shape PEG, and a 4-arm branchedPEG, 6-arm branched PEG or 8-arm branched PEG, and the like.

In one embodiment of the invention, the PEG is a linear polyethyleneglycol residue having the structure shown in Formula II or III:

wherein Y is a terminal group selected from the group consisting of:C1-C6 alkoxy group, hydroxyl, carboxyl group, succinimide carbonategroup, succinimide acetate group, succinimide propionate group, and asuccinyl group. Imine succinate group, succinimide group, dithiopyridylgroup, propionic acid group, aldehyde group, thiol ester group,acryloxy, acrylic acid group, azido group, glutaric acid group, alkynyl,p-nitrogen one of a phenyl carbonate group, silane, and carboxymethyl; pand q are independently selected from an integer of from 1 to 1400,preferably from 1 to 1200, more preferably from 200 to 1200.

In one embodiment of the invention, in Formula II, said Y is methoxy.

In one embodiment of the invention, the PEG is a Y- or U-shapepolyethylene glycol residue having the structure shown in Formula IV orV:

Wherein Y is a terminal group having the above definition of the presentinvention, i and k are independently selected from an integer from 1 to1200, preferably from an integer from 1 to 600, more preferably from 100to 600.

In one embodiment of the invention, in Formula IV and/or V, said Y ismethoxy.

In one embodiment of the invention, the PEG is a multi-branchedpolyethylene glycol residue having the structure shown in Formula VI:

Wherein Y is an terminal group having the above definition of thepresent invention, n is an integer from 1 to 800, preferably an integerfrom 1 to 400, more preferably an integer from 60 to 400, 1 is aninteger from 0 to 7, and j is an integer from 1 to 8; preferably,3≤j+1≤8, R is a core molecule of a multi-branched polyethylene glycol,and R is selected from the group consisting of: pentaerythritol,oligo-pentaerythritol, methyl glucoside, sucrose, diethylene glycol,propylene glycol, glycerin, and polyglycerol residues.

In one embodiment of the invention, in Formula VI, said Y is methoxy.

In one embodiment of the invention, the 1 is zero.

In a preferred embodiment of the invention, the PEG is a multi-branchedpolyethylene glycol residue, and the polyethylene glycol maleimidederivative has the following structure:

In one embodiment of the invention, the polyethylene glycol maleimidederivative has the following structure:

Preferably, in formula VIII, j=8, the PEG is an eight-arm polyethyleneglycol residue, and the polyethylene glycol maleimide derivative is aneight-arm polyethylene glycol maleimide derivative.

In a specific embodiment of the present invention, in formula VIII, j=8,the R has the structure represented by formula IX or X:

In a preferred embodiment of the invention, the polyethylene glycolmaleimide derivative has the following structure:

In one embodiment of the present invention, the PEG may have a molecularweight of 1-80 KDa, such as 1-10 KDa (specifically 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 KDa), 10-80 KDa (specifically 10, 15, 20, 25, 30, 35, 40,45, 50, 60, 70 or 80 KDa); preferably 10-50 KDa.

In a preferred embodiment of the invention, the PEG has a molecularweight of 10 KDa, 20 KDa or 40 KDa.

In the present invention, the polymerization inhibitor is a substancewhich converts a primary radical or a chain radical into a stablemolecule or to form a stable radical which is low in activity and whichis insufficient for the polymerization to proceed. It will be understoodby those skilled in the art that the polymerization inhibitor can beclassified into the following categories based on the substituent(s):free radicals, phenols, quinones, aromatic amines, nitro compounds, anitroso compound, sulfur-containing compounds, inorganic compounds, ororganometallic compounds, oxygens, etc. (see Xiao Weidong, He Benqiao,He Peixin. Chemical additives for polymer materials. Beijing: ChemicalIndustry Press, 2003: 331-341″).

In one embodiment of the present invention, the polymerization inhibitoris selected from one or a combination of two or more of a radicalpolymerization inhibitor, a phenolic polymerization inhibitor, aninorganic compound polymerization inhibitor, and an organometalliccompound polymerization inhibitor.

In one embodiment of the present invention, the radical polymerizationinhibitor includes, but not limited to, one or more of1,1-diphenyl-2-picrylyl radical (1,1-Diphenyl-2-picrylhydrazyl, DPPH),nitroxide radicals such as 4,4′-dimethoxydiphenylnitroxyl radical(4,4′-dimethoxydiphenyl oxynitride, DMDPN), 4,4′-dinitrodiphenylnitroxylradical (4,4′-dinitrodiphenyl oxynitride), di-tert-butyl nitroxylradical (di-tert-butyl oxynitride, DTBNO),2,2,6,6-tetramethyl-4-hydroxypiperidine nitroxyl radical(2,2,6,6-tetramethyl-4-hydroxypiperidine oxynitride, TEMPO), benzoylperoxide (BPO).

In a specific embodiment of the invention, the radical polymerizationinhibitor is DPPH.

In one embodiment of the present invention, the phenolic polymerizationinhibitor includes, but not limited to, one or more of hydroquinone,p-tert-butyl phenol, methyl hydroquinone, p-tert-butyl catechol,p-hydroxybenzene. Methyl ether, 2-tert-butyl hydroquinone,2,5-di-tert-butyl hydroquinone, 2,6-di-tert-butyl-4-methylphenol (BHT),4,4′-dihydroxybiphenyl, pyrogallol and bisphenol A.

In a preferred embodiment of the invention, the phenolic polymerizationinhibitor is hydroquinone and/or p-tert-butyl phenol.

In one embodiment of the present invention, the inorganic compoundpolymerization inhibitor includes, but is not limited to, one or more offerric chloride, cuprous chloride, copper chloride, copper sulfate,titanium trichloride, titanium chloride, sodium sulfate, sodium sulfideand ammonium thiocyanate.

In a specific embodiment of the invention, the inorganic compoundinhibitor is copper chloride.

In one embodiment of the invention, the organometallic compoundpolymerization inhibitor includes, but not limited to, one or more ofcopper naphthenate, chromium acetate, nickel acetate, copper dimethyldithiocarbamate, and copper dit-butyl dithiocarbamate.

In a specific embodiment of the invention, the organometallic compoundpolymerization inhibitor is nickel acetate.

The inventors of the present invention have experimentally found thatthe combination of phenolic polymerization inhibitors (such ashydroquinone and p-t-butyl phenol) and polyethylene glycol maleimidederivatives (especially eight-arm polyethylene glycol maleimidederivatives) can significantly enhance the stability, reduce thephotosensitivity, and extend the shelf life and storage period of thederivatives, and the effect is better than other combinations ofpolyethylene glycol maleimide derivatives with other polymerizationinhibitors.

In a preferred embodiment of the present invention, in the composition,the polymerization inhibitor is a phenolic polymerization inhibitor.Preferably the polymerization inhibitor is selected from: one or acombination of two or more of hydroquinone, p-t-butyl phenol, methylhydroquinone, tert-butyl catechol, 2-tert-butyl hydroquinone,2,5-di-tert-butyl hydroquinone, 4-methoxyphenol,2,6-di-tert-butyl-4-methylphenol, 4,4′-dihydroxybiphenyl, pyrogallol andbisphenol A. More preferably, the polymerization inhibitor ishydroquinone or p-tert-butyl-phenol.

In a preferred embodiment of the invention, in the composition, thepolyethylene glycol maleimide derivative has the structure of the aboveformula VIII, wherein, preferably, j=8, more preferably, the R has thestructure of the above formula IX or X, and most preferably, thepolyethylene glycol maleimide derivative has the structure of the aboveformula XI; preferably, the molecular weight of the PEG is 1-80 KDa, forexample, 1-10 KDa (specifically 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 KDa),10-80 KDa (specifically 10, 15, 20, 25, 30, 35, 40, 45, 50 60, 70 or 80KDa), more preferably 10-50 KDa, most preferably 10 KDa, 20 KDa and 40KDa; in the composition, the polymerization inhibitor is a phenolicpolymerization inhibitor, and preferably is selected from: one or moreof hydroquinone, p-tert-butyl-phenol, methyl hydroquinone, p-tert-butylcatechol, 2-tert-butyl hydroquinone, 2,5-di-tert-butyl hydroquinone,4-methoxyphenol, 2,6-di-tert-butyl-4-methylphenol,4,4′-dihydroxybiphenyl, pyrogallol and bisphenol A, more preferably thepolymerization inhibitor is hydroquinone or tert-butyl Phenol; in thecomposition, the mass ratio of the phenolic polymerization inhibitor andthe polyethylene glycol maleimide derivative may be from 0.1 μg to 10mg:1 g, such as from 0.1 to 100 μg (eg, 0.1 μg, 1 μg, 5 μg:1 g, 10 μg:1g, 20 μg:1 g, 30 μg:1 g, 40 μg:1 g, 50 μg:1 g, 60 μg:1 g, 70 μg:1 g, 80μg:1 g, 90 μg:1 g, 100 μg:1 g), 100 μg-1 mg:1 g (eg, 100 μg:1 g, 200μg:1 g, 300 μg:1 g, 400 μg:1 g, 500 μg:1 g, 600 μg:1 g, 700 μg:1 g, 800μg:1 g, 900 μg:1 g, 1 mg:1 g), 1-10 mg:1 g (eg, 1 mg:1 g, 2 mg:1 g, 3mg:1 g, 4 mg:1 g, 5 mg:1 g, 6 mg:1 g, 7 mg:1 g, 8 mg:1 g, 9 mg:1 g, 10mg:1 g).

In a more preferred embodiment of the invention, in the composition, thepolymerization inhibitor is a phenolic polymerization inhibitor,preferably is hydroquinone or p-tert-butyl-phenol, the polyethyleneglycol maleimide derivative has the structure of the above formula VIIIof the present invention, and the mass ratio of the phenolicpolymerization inhibitor to the polyethylene glycol maleimide derivativemay be from 1 μg to 1 mg:1 g.

Another aspect of the invention also provides the use of a compositionas described above for the preparation of a pharmaceutical conjugate.

Another aspect of the present invention also provides a pharmaceuticalcomposition comprising a polymerization inhibitor and a conjugate of apolyethylene glycol maleimide derivative of the present invention and adrug.

Preferably, the polymerization inhibitor is a phenolic polymerizationinhibitor, more preferably is selected from: one or more ofhydroquinone, p-t-butyl phenol, methyl hydroquinone, p-tert-butylcatechol, 2-tert-butyl hydroquinone, 2,5-di-tert-butyl hydroquinone,4-methoxyphenol, 2,6-di-tert-butyl-4-methylphenol,4,4′-dihydroxybiphenyl, pyrogallol and bisphenol A, most preferably thepolymerization inhibitor is hydroquinone and/or p-tert-butyl-phenol.

In one embodiment of the invention, the drug is selected from one ormore of amino acids, polypeptides, proteins, sugars, organic acids,alkaloids, flavonoids, terpenoids, terpenoids, phenylpropanoid phenols,steroids, and quinones.

In a specific embodiment of the invention, the drug is a polypeptide orprotein drug.

In a specific embodiment of the invention, the polypeptide or proteindrug contains a free sulfhydryl group.

In another embodiment of the invention, a sulfhydryl group is introducedat a specific site in the polypeptide or protein drug.

In a specific embodiment of the present invention, the polypeptide orprotein drug includes, but is not limited to, a polypeptide hormone, apolypeptide cell growth regulator, other biochemical drugs containing apolypeptide component, a protein hormone, a plasma protein, and aprotein cell growth regulators, mucins, lectins, antibodies and enzymessuch as a urea oxidase.

In one embodiment of the invention, the polypeptide hormones include,but are not limited to, pituitary polypeptide hormones (such ascorticotropin, melatonin, lipolysis hormone, oxytocin, etc.),hypothalamic hormones (such as thyrotropin releasing hormone), auxininhibitory hormone, gonadotropin-releasing hormone, etc.), thyroidhormone (such as parathyroid hormone, calcitonin, pancreas hormone:glucagon, pancreatic spasmolytic polypeptide, etc.), gastrointestinalhormones (such as gastrin, cholecystokinin-trypsin, vasoactiveintestinal peptide, etc.) and thymus hormones (such as thymosin, thymusserum factor, etc.).

In one embodiment of the invention, the polypeptide cell growthregulators include, but are not limited to, epidermal growth factor,transfer factor, and atrial natriuretic peptide.

In one embodiment of the present invention, the other biochemical drugscontaining a polypeptide component include, but are not limited to,Ossotide, Ocustrilla Extracti Oculi, soleosery, Antaisu, Fuxuening,brain aminopeptide, bee venom, snake venom, embryonic hormone, cofactor,neurotrophic factor, placental extract, pollen extract, spleenhydrolysate, liver hydrolysate and cardiac hormone.

In one embodiment of the present invention, the protein hormoneincludes, but is not limited to, pituitary protein hormones (such asauxin, prolactin, thyrotropin, luteinizing hormone, follicle stimulatinghormone, etc.), gonadotropin (such as human chorionic gonadotropin,serotonin, etc.) and insulin and other protein hormones such as relaxin.

In one embodiment of the invention, the plasma protein includes, but isnot limited to, albumin, plasminogen, plasma fibronectin,immunoglobulin, fibrinogen, and coagulation factors.

In one embodiment of the invention, the proteinaceous cell growthregulators include, but are not limited to, interleukin (IL-1 to IL-38),colony stimulating factor (eg, granulocyte colony stimulating factor(G-CSF)), macrophage colony-stimulating factor (M-CSF), granulocyte andmacrophage colony-stimulating factor (GM-CSF), multi-colony stimulator(Multi-CSF), stem cell factor (SCF), erythropoietin (EPO)), interferon(α, β, γ), growth factors (such as epidermal growth factor,platelet-derived growth factor, fibroblast growth factor, hepatocytegrowth factor, insulin-like growth factor, nerve growth factor,platelet-derived) Endothelial growth factor, vascular endothelial growthfactor, transforming growth factor-α, etc., tumor necrosis factor,tissue plasminogen activator and erythropoietin.

In one embodiment of the invention, the mucin comprises, but is notlimited to, gastric factor, collagen, a basic protein, and a proteaseinhibitor such as a trypsin inhibitor.

In one embodiment of the invention, the lectin includes, but is notlimited to, concanavalin, wheat germ, peanut agglutinin, soybean lectin,and the like.

In one embodiment of the invention, the antibodies include, but are notlimited to, anti-CD20 antibodies, antibodies against the EGFR family(eg, anti-EGFR antibodies, anti-HER-2 antibodies), and anti-VEGF/VEGFRantibodies, and the like.

In one embodiment of the invention, the enzyme includes, but is notlimited to, L-asparaginase, glutaminase, urokinase, neuraminidase,superoxide dismutase, and the like.

Another aspect of the invention also provides a pharmaceutical conjugateprepared as described above.

Another aspect of the invention also provides the use of thepolymerization inhibitor in the preparation of the above compositions,pharmaceutical combinations and pharmaceutical compositions.

Preferably, in the above application, the polymerization inhibitor is aphenolic polymerization inhibitor, more preferably, the polymerizationinhibitor is selected from: one or more of hydroquinone, p-t-butylphenol, methyl hydroquinone, p-tert-butyl catechol, 2-tert-butylhydroquinone, 2,5-di-tert-butyl hydroquinone, 4-methoxyphenol,2,6-di-tert-butyl-4-methylphenol, 4,4′-dihydroxybiphenyl, pyrogallol andbisphenol A. Most preferably, the polymerization inhibitor ishydroquinone and/or p-tert-butyl phenol.

The invention provides a composition of a polyethylene glycol maleimidederivative and a polymerization inhibitor, in particular a combinationof an eight-arm polyethylene glycol maleimide derivative and a phenolicpolymerization inhibitor (such as hydroquinone and p-tert-butyl-phenol),wherein the structure and content of the polymerization inhibitor couldbe selected reasonably, and the stability of the polyethylene glycolmaleimide derivative can be greatly enhanced, thereby effectivelyavoided adverse effect of gelation during the periods of transportationand storage, prolong the pot life and storage period of the product, andis effective for preparing drug conjugates, especially polypeptide(polypeptidic) and protein (proteinaceous) drug conjugates.

DRAWINGS

FIG. 1 shows the GPC pattern of 8ARM(TP)-PEG-MAL-40K with apolymerization inhibitor added after 2 days irradiation.

FIG. 2 shows the effect of the type and concentration or content of thepolymerization inhibitor on the time of 8ARM(TP)-PEG-MAL-40K before thecomplete deterioration.

FIG. 3 shows the effect of the type and concentration or content of thepolymerization inhibitor on the time of 8ARM(TP)-PEG-MAL-20K before thecomplete deterioration.

FIG. 4 shows the effect of the type and concentration or content of thepolymerization inhibitor on the time of 8ARM(TP)-PEG-MAL-10K before thecomplete deterioration.

FIGS. 5A and 5B show the GPC detection spectrum in Example 5, whereinFIG. 5A is the GPC detection spectrum with hydroquinone, and FIG. 5B isthe GPC detection spectrum without hydroquinone.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used in thepresent invention have the same meaning as commonly understood by one ofordinary skill in the art to which this invention pertains.

“Alkyl” refers to a hydrocarbon chain radical that is linear or branchedand free of unsaturated bonds. The C₁-C₆ alkyl means an alkyl having 1to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, tert-amyl, n-hexyl,isohexyl and the like.

“Alkoxy” means a substituent formed by substituting the hydrogen inhydroxy group with an alkyl group, and C1-C6 alkoxy group means analkoxy group having 1 to 6 carbon atoms, such as methoxy or ethoxy,propoxy, butoxy, and the like.

“Cycloalkyl” means an alicyclic hydrocarbon, such as those containing 1to 4 monocyclic and/or fused rings. It may contain 3 to 18 carbon atoms,preferably 3 to 10 carbon atoms, such as cyclopropyl, cyclohexyl oradamantyl and the like. C3-C6 cycloalkyl in the present invention meansa cycloalkyl having 3 to 6 carbon atoms, such as cyclopropyl,cyclobutyl, cyclopentyl and cyclohexyl.

In addition, some specific groups and their chemical structures involvedin the present invention correspond to the following: hydroxyl group,—OH;

(wherein Q₁ may be an alkyl group or a heterocyclic group such asmethyl, ethyl, n-propyl, tert-butyl, pyridyl (such as

etc.);

glutarate, such as

(wherein Q₂ may be the same or different alkyl or alkoxy group, such asmethyl, ethyl, propyl, butyl, pentyl, methoxy, ethoxy, propyl Oxyl,butoxy, etc., preferably, Q₂ is methyl, ethyl, n-propyl, methoxy,ethoxy, n-propoxy, etc.);

In the definition of a linking group in the present invention, the“combination” means a group formed by linking two or more of the listedlinking groups by a chemical bond. For example, the combination of—(CH₂)_(r)— and —(CH₂)_(r)NHCO— may be —(CH₂)_(r)NHCO(CH₂)_(r)— andspecifically, the combination of —CH₂— and —CH₂CH₂NHCO— may be—CH₂CH₂NHCOCH₂—, —CH₂CH₂CH₂NHCO—. The “combination” is used to definethe chemical structure of the linking group, and does not involve thepreparation steps, the order of linking groups in the combination, etc.

The polyethylene glycol maleimide derivative described in the presentinvention is a polyethylene glycol to which a maleimide group isattached, as shown in formula I of the present invention, specificallyas shown in formula XI; the linkage can be achieved by a covalent bondor by a linking group, and the reaction for achieving the linkage iswell known to those skilled in the art, which is not specificallydefined in the present invention.

The “composition of a polyethylene glycol maleimide derivative and apolymerization inhibitor” described in the present invention may furthercontain other components, such as ultraviolet absorbers (UV-P) toimprove product quality or function. Those skilled in the art can addother components according to actual needs. Moisture, impurities and thelike are inevitably involved in the process of production,transportation or storage of the polyethylene glycol maleimidederivatives and the compositions thereof and these substances are notspecifically defined in the present invention.

The technical solutions of the present invention will be describedclearly and completely with reference to the embodiments of the presentinvention. It is obvious that these embodiments are only a part of thepossible embodiments of the present invention, and not all of theembodiments.

All other embodiments obtained by a person of ordinary skill in the artbased on the embodiments of the present invention without involvement ofinventive step are within the scope of the present invention.

The compounds used in the present invention are either commerciallyavailable or can be prepared according to the disclosed preparationmethods, which are not meant to limit the scope of the invention.

The polyethylene glycol derivative used in the examples was supplied byJENKEM Technology Co., Ltd. (Beijing). All others are commerciallyavailable reagents.

Example 1

Hydroquinone, DPPH, nickel acetate, copper chloride and was respectivelyobtained and mixed with 8ARM(TP)-PEG-MAL-40K (250 mg) to formcomposition powders, wherein hydroquinone, DPPH, nickel acetate andcopper chloride were all at 100 ppm.

The above composition powders were separately placed in a watch glassand placed in a light box at a constant temperature (2750 Lux, 18.5° C.)for two days, and the GPC results are shown in FIG. 1 .

According to FIG. 1 , the GPC concentration or content comparisonresults after 2 days of irradiation of 8ARM(TP)-PEG-MAL-40K (to which apolymerization inhibitor was added) are shown in Table 1.

TABLE 1 GPC concentration or content after 2 days irradiation of8ARM(TP)-PEG-MAL-40K with polymerization inhibitor added Addition of apolymerization inhibitor nickel copper hydroquinone DPPH acetatechloride 8ARM(TP)-P 83.9 77.3 67.9 54.3 EG-MAL-40K purity(%)

As shown in Table 1, among the above four polymerization inhibitors,with the addition of hydroquinone, 8ARM(TP)-PEG-MAL-40K had the lowestdegree of purity reduction, with the best inhibiting effect.

Example 2

Different types and concentrations or content of additives were obtainedand mixed with 8ARM(TP)-PEG-MAL-40K (250 mg) to form compositionpowders: hydroquinone, p-tert-butyl-phenol, a mixture of hydroquinoneand UV absorber (hydroquinone & UV-P, the mass ratio is 1:1), DPPH,nickel acetate, copper chloride, wherein the concentrations or contentof each additive were at 1, 20, 100, 400 and 1000 ppm.

The above composition powders were separately placed in a watch glassand placed in a light box at a constant temperature (2750 Lux, 18.5° C.)to record the time required before complete deterioration (i.e., when agel insoluble in methanol occurred), and the result was as shown in FIG.2 (Note: If no additives were added, 8ARM(TP)-PEG-MAL-40K werecompletely deteriorated after two days of irradiation).

It can be seen from FIG. 2 that the effect of phenolic polymerizationinhibitors such as hydroquinone and p-tert-butyl-phenol are better thanthe other polymerization inhibitors, and the mixture of hydroquinone andUV-P has the similar effect as that of hydroquinone alone.

Example 3

Composition powders containing different concentrations or content ofhydroquinone, DPPH, nickel acetate, copper chloride and8ARM(TP)-PEG-MAL-20K (250 mg), were separately taken, wherein theconcentration or content of each polymerization inhibitor were at 1, 20,100, 400, 1000 ppm.

The above composition powders were separately placed in a watch glassand placed in a light box at a constant temperature (2750 Lux, 18.5° C.)to record the time required before complete deterioration (i.e., when agel insoluble in methanol occurred), and the result was as shown in FIG.3 (Note: If no inhibitor is added, 8ARM(TP)-PEG-MAL-20K would completelydeteriorate after two days of irradiation).

Example 4

A composition powder containing different concentrations or content ofhydroquinone, DPPH, nickel acetate, copper chloride and8ARM(TP)-PEG-MAL-10K (250 mg) was separately taken, wherein theconcentration or content of each polymerization inhibitor was at 1, 20,100, 400, 1000 ppm.

The above composition powders were separately placed in a watch glassand placed in a light box at a constant temperature (2750 Lux, 18.5° C.)to record the time required before complete deterioration (i.e., a gelinsoluble in methanol occurred), and the result was as Shown in FIG. 4(Note: If no inhibitor is added, 8ARM(TP)-PEG-MAL-10K would completelydeteriorate after one day of irradiation).

It can be seen from Examples 2-4 that the above polymerizationinhibitors, particularly hydroquinone, have a good stabilizing effect onpolyethylene glycol maleimide derivatives of different molecularweights.

The 8ARM(TP)-PEG-MAL used in the embodiment of the present invention hasthe following structure:

Enhanced stability, prolonged pot life and storage period could beachieved all the same for the maleimide derivative of polyethyleneglycol of other configurations and molecular weights, when mixed withthe polymerization inhibitor in the examples of the present invention.Those experimental data are not shown herein.

Example 5

1 g of 8ARM(TP)-MAL-40K and 10 mg of hydroquinone were weighed and putinto 50 ml single mouth round-bottomed flask and dissolved by 10 ml ofdichloromethane. In addition, 1 g of 8ARM(TP)-MAL-40K were weighed andput into a 50 ml single mouth round-bottomed flask and dissolved by 10ml dichloromethane. After the two systems were placed under anincandescent lamp for 48 hours, 40 mg thiosalicylic acid was addedrespectively. The reaction lasted for two hours. Samples were taken forGPC test, the GPC test spectrum is shown in FIGS. 5A-5B, and thecorresponding spectrum data are shown in Table 2 and Table 3. The GPCtest results in Table 4 show that the purity of pharmaceutical conjugatein the reaction system with hydroquinone is 89.8%, and that in thereaction system without hydroquinone is 87%, and the percentages of thehigh molecular weight impurity and the low molecular weight impurity areless than those in the system without hydroquinone.

TABLE 2 Peak Retention number time Area Height Concentration 1 13.98640155 1083 5.789 2 14.877 622103 16701 89.819 3 16.281 30363 962 4.384Total 692621 18747

TABLE 3 Peak Retention number time Area Height Concentration 1 13.97577989 1972 7.306 2 14.882 928480 25133 86.981 3 16.296 60980 1535 5.713Total 1067449 28640

TABLE 4 Main High molecular Low molecular product weight impurity weightimpurity GPC test (%) (%) (%) With Hydroquinone 89.8 5.8 4.4 withoutHydroquinone 87 7.3 5.7

The above results show that the addition of polymerization inhibitor(such as hydroquinone) in the reaction system can improve the purity ofpharmaceutical conjugate products.

The above is only the preferred embodiment of the present invention, andis not intended to limit the present invention. Any modifications,equivalent substitutions, etc., which are within the spirit andprinciples of the present invention, should be included in the scope ofthe present invention.

The invention claimed is:
 1. A pharmaceutical conjugate, comprising acomposition of a polyethylene glycol maleimide derivative and apolymerization inhibitor and a drug, wherein the mass ratio of thepolymerization inhibitor to the polyethylene glycol maleimide derivativeis ≥0.1 μg:1 g, the polyethylene glycol maleimide derivative contains atleast one terminal maleimide group.
 2. The pharmaceutical conjugateaccording to claim 1, wherein said polymerization inhibitor is selectedfrom one or a combination of two or more of a radical polymerizationinhibitor, a phenolic polymerization inhibitor, an inorganic compoundpolymerization inhibitor, and an organometallic compound polymerizationinhibitor.
 3. The pharmaceutical conjugate according to claim 1, whereinthe mass ratio of the polymerization inhibitor to the polyethyleneglycol maleimide derivative is from 0.1 μg to 10 mg:1 g.
 4. Thepharmaceutical conjugate according to claim 1, wherein the polyethyleneglycol maleimide derivative has the following structure: PEG-X-MAL (I),wherein PEG is polyethylene glycol residue, X is a linking group of PEGand MAL, and is selected from: one or a combination of two or more of—(CH₂)_(r)—,

—(CH₂)_(r)O—, —(CH₂)_(r)CO—, —(CH₂)_(r)NH—, —(CH₂)_(r)CONH—,—(CH₂)_(r)NHCO—, —(CH₂)_(r)S—, —(CH₂)_(r)COO— and —(CH₂)_(r)OCO—, r isan integer from 0 to 10, MAL is a maleimide group

R₁ and R₂ are independently selected from: —H, a C1-6 alkyl group, aC1-6 alkoxy group, a C3-6 cycloalkyl group, and a C4-10 alkylenecycloalkyl group.
 5. The pharmaceutical conjugate according to claim 4,wherein said X is selected from the group consisting of: a single bond,—CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH(CH₃)—, —CH₂CH(CH₃)—, —CH₂CH₂CH(CH₃)—,—(CH₂)_(r)O—, —(CH₂)_(r)CO—, —(CH₂)_(r)NH—, —(CH₂)_(r)CONH— and—(CH₂)_(r)NHCO—; and/or, the r is an integer from 0 to
 5. 6. Thepharmaceutical conjugate according to claim 4, wherein said PEG is alinear polyethylene glycol residue having a structure shown in formulaII or III:

wherein p and q are independently selected from the group consisting ofan integer of 1-2400; or, the PEG is a Y-shape or U-shape polyethyleneglycol residue having a structure of formula IV or V:

wherein i and k are independently selected from an integer of 1-1200; orPEG is a multi-branched polyethylene glycol residue having the structureshown in formula VI:

wherein n is an integer from 1 to 800, 1 is an integer from 0 to 7, andj is an integer from 1 to 8, R is a core molecule of a multi-branchedpolyethylene glycol, and R is selected from the group consisting of:pentaerythritol, oligo-pentaerythritol, methyl glucoside, sucrose,diethylene glycol, propylene glycol, glycerol, and polyglycerolresidues; wherein Y is a terminal group selected from the groupconsisting of: C1-C6 alkoxy group, hydroxyl, carboxyl group, succinimidecarbonate group, succinimide acetate group, succinimide propionategroup, and succinimide succinate group, succinimide group, dithiopyridylgroup, propionic acid group, aldehyde group, thiol ester group, acryloxygroup, acrylic acid group, azido group, glutaric acid group, alkynyl,4-nitrophenyl carbonate, silane, and carboxymethyl.
 7. Thepharmaceutical conjugate according to claim 6, wherein said polyethyleneglycol maleimide derivative has the following structure:

and, j=8, PEG is an eight-arm polyethylene glycol residue.
 8. Thepharmaceutical conjugate of claim 7 wherein R has the structure offormula IX or X:


9. The pharmaceutical conjugate according to claim 6, wherein PEG has amolecular weight of from 1 to 80 kDa.
 10. The pharmaceutical conjugateaccording to claim 2, wherein the radical polymerization inhibitor isselected from one or more of 1,1-diphenyl-2-trinitrophenylhydrazine,4,4′-dimethoxydiphenyl oxynitride, 4,4′-dinitrodiphenyl oxynitride,di-tert-butyl oxynitride, 2,2,6,6-tetramethyl-4-hydroxyl piperidineoxynitride and benzoyl peroxide; and/or said phenolic polymerizationinhibitor is selected from one or more of: hydroquinone, methylhydroquinone, p-tert-butylcatechol, 2-tert-butyl hydroquinone,4-methoxyphenol, 2,5-di-tert-butyl hydroquinone,2,6-di-tert-butyl-4-methylphenol, 4,4′-dihydroxybiphenyl, pyrogallol andbisphenol A; and/or said inorganic compound polymerization inhibitor isselected from one or more of: ferric chloride, cuprous chloride, copperchloride, copper sulfate, titanium trichloride, titanium chloride,sodium sulfate, sodium sulfide and ammonium thiocyanate; and/or saidorganometallic compound polymerization inhibitor is selected from one ormore of: naphthenic acid copper, chromium acetate, nickel acetate,copper dimethyldithioformate, and copper dit-butyldithiocarbamate. 11.The pharmaceutical conjugate according to claim 1, wherein the radicalpolymerization inhibitor is 1,1-diphenyl-2-trinitrophenylhydrazine;and/or, the phenolic polymerization inhibitor is hydroquinone orp-tert-butyl phenol; and/or the inorganic compound polymerizationinhibitor is copper chloride; and/or the organometallic compoundpolymerization inhibitor is nickel acetate.
 12. The pharmaceuticalconjugate according to claim 1, wherein the polymerization inhibitor isa phenolic polymerization inhibitor.
 13. The pharmaceutical conjugateaccording to claim 1, wherein the polymerization inhibitor is selectedfrom one or more of: hydroquinone, methyl hydroquinone,p-tert-butylcatechol, 2-tert-butyl hydroquinone, 4-methoxyphenol,2,5-di-tert-butyl hydroquinone, 2,6-di-tert-butyl-4-methylphenol,4,4′-dihydroxybiphenyl, pyrogallol and bisphenol A.
 14. Thepharmaceutical conjugate according to claim 1, wherein thepolymerization inhibitor is hydroquinone or p-tert-butyl-phenol.
 15. Thepharmaceutical conjugate according to claim 1, wherein the drug isselected from the group consisting of amino acids, polypeptides,proteins, sugars, organic acids, alkaloids, flavonoids, terpenoids,terpenoids, phenylpropanoid phenols, steroids and steroids.
 16. Thepharmaceutical conjugate according to claim 15, wherein the drug is apolypeptide or protein drug.
 17. The pharmaceutical conjugate accordingto claim 16, wherein the polypeptide or protein drug contains freesulfhydryl groups; and/or, a sulfhydryl group is introduced at aspecific site in the polypeptide or protein drug.